兰州大学机构库 >生命科学学院
SIRT6 截短体的构建、表达、纯化及酶活检测
Alternative TitleConstruction, expression, purification and enzyme activity determination of truncated protein of SIRT6
王承祥
Thesis Advisor张健 ; 沈剑敏
2016-05-18
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name学士
KeywordSIRT6 截短体 蛋白表达 纯化 酶活检测
AbstractSIRT6 属于Sirtuins家族,主要功能是通过催化细胞内相关的蛋白进行去乙酰化和单腺苷二磷酸核糖基化参与维持基因组的稳定性,对DNA修复、缓解炎症、葡萄糖代谢及脂类代谢都有重要意义,与糖尿病、癌症、心脏疾病和衰老等疾病密切相关。构建SIRT6 的截短体有助于研究SIRT6 的去乙酰化酶活性调节机制,进行蛋白结晶以及研究其结构与功能的对应关系,为开发相应的激动剂或抑制剂提供基础理论依据。我们通过构建SIRT6 基因的截短体表达重组质粒,在大肠杆菌中进行诱导表达,收集菌体进行纯化,纯化方法包括镍柱亲和层析、分子排阻层析和离子交换层析,并获得纯化好的蛋白进行酶活性检测。可以发现随着从C端往N端截短,SIRT6 去乙酰化酶活性逐渐降低,并且趋势呈非线性,我们推测C端的残基可能对调节酶的催化活性或稳定蛋白构象有重要意义。
Other AbstractSIRT6 belongs to the Sirtuins family. It participates in maintenance of genome stability through catalyzing deacetylation and mono-ADP-ribosylation on intracellular proteins. SIRT6 has both enzyme activities of deacetylation and ADP-ribosy transformation and influence on DNA repair, inflammation, glucose and lipid metabolism, which are closely related to cancer, heart disease, diabetes, obesity, aging and other diseases. This promotes the research on the truncated proteins on SIRT6, which contributes to explore the mechanism of reactive regulation of deacetylation of SIRT6, crystallization of protein, studying the relations between structure and function of SIRT6, and providing theory basis for developing activators and inhibitors. First of all, we constructed plasmids of truncated protein of SIRT6 to induce expression in E.coli. Next, we collected bacterial suspension to purify the protein. We used nickel affinity chromatography, molecular-exclusion chromatography and ion exchange chromatography. Finally, the enzyme activity of proteins purified was detected by SIRT6 Fluorescent Screening Assay Kit. The result showed that the enzyme activity decreases nonlinearly with the truncation of protein from C-terminal to N-terminal. We speculate that the amino acid residue in C-terminal may have great effects on enzyme activity and the maintenance of protein conformation.
URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/221857
Collection生命科学学院
Recommended Citation
GB/T 7714
王承祥. SIRT6 截短体的构建、表达、纯化及酶活检测[D]. 兰州. 兰州大学,2016.
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